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Home , Chromosome 1, RAD23B, RAD51, UBE2I

GENOMICS 36, 271 –279 (1996) ARTICLE NO. 0462

UBL1, a Human -like Associating with Human RAD51/RAD52

ZHIYUAN SHEN,* PAIGE E. PARDINGTON-PURTYMUN,* JARMON C. COMEAUX,* ,1 ROBERT K. MOYZIS,† AND DAVID J. CHEN* *DNA Damage and Repair Group, Life Sciences Division, and †Center for Human Studies, Los Alamos National Laboratories, Los Alamos, New Mexico 87545

Received February 16, 1996; accepted June 13, 1996

RAD55, and RAD57 (Adzuma et al., 1984; Alani et al., The RAD51/RAD52-dependent DNA repair pathway 1989; Basile et al., 1992; Emery et al., 1991; Kans and is involved in DNA recombination and DNA double- Mortimer, 1991; Lovett, 1994; Schild et al., 1983; Shi- strand break repair in yeast. Although many proteins nohara et al., 1992; Zheng et al., 1993). It is known in the RAD51/RAD52-dependent DNA repair pathway that RAD51 is a RecA-like protein that has DNA-bind- have been identified in yeast, a novel protein(s) that ing, ATP-binding, and DNA strand-exchange activities, functions with RAD51/RAD52 may also exist in hu- while RAD54 contains regions homologous to DNA hel- mans. Using a yeast two-hybrid system, we have identi- icase. Interactions among RAD52, RAD51, RAD55, and fied a 12-kDa protein that associates with the human RAD57 have been identified (Firmenich et al., 1995; RAD51 and RAD52 proteins. This protein shares sig- Hays et al., 1995; Shinohara et al., 1992; Smith and nificant with the yeast protein SMT3, which functionally associates with the yeast mi- Rothstein, 1995). RAD52 and RAD54 are highly ex- tosis fidelity protein MIF2. It also shares moderate ho- pressed during yeast (Cole et al., 1989). Yeast mology with ubiquitin and several other proteins, in- RAD51 and RAD53 also have cell-cycle-dependent ex- cluding the N-terminus of the RAD23 protein and a pression (Basile et al., 1992; Zheng et al., 1993), and ubiquitin cross reacting protein. Therefore, the is RAD53 also participates in cell cycle control (Sanchez tentatively designated UBL1 for ubiquitin-like 1. The et al., 1996). UBL1 mRNA is expressed in many human tissues, most In humans, only RAD51 and RAD52 have been re- highly in testis. The UBL1 gene is mapped to chromo- ported (Muris et al., 1994; Shen et al., 1995; Shinohara some 2q32.2–q33, and a related sequence may be lo- et al., 1993). The human RAD51 protein has activities cated on 1q23–q25. ᭧ 1996 Academic Press, Inc. similar to those of yeast RAD51, including DNA bind- ing and a specific interaction with RAD52 protein (Ben- son et al., 1994; Shen et al., 1996a). Also, we have found INTRODUCTION that human RAD52 protein self-associates (Shen et al., 1996b). More recently, RAD51 has been shown to accu- DNA double-strand break (DSB)2 is one of the most mulate in the , indicating important forms of DNA damage caused by ionizing involvement of RAD51 in meiosis and chromosome re- radiation. Efficient repair of DSB is essential for the combination (Haaf et al., 1995). Human RAD52 overex- cell to recover from radiation damage. In yeast, the pression in monkey cells enhances their resistance to RAD52 epistasis group encode proteins involved radiation and increases the frequency of homologous in DSB repair and recombination (Friedberg et al., recombination (Park, 1995). However, little is known 1991). Several yeast genes in the RAD52-dependent about how these interactions might fit into the context repair pathway have been cloned. These include of DSB repair. RAD50, RAD51, RAD52, RAD53 (SPK1), RAD54, The importance of DSB repair factors is not limited to their roles in modulating radiation sensitivity, but Sequence data from this article have been deposited with the Gen- includes their roles in DNA recombination, in repair of Bank/EMBL Data Libraries under Accession No. U38784. alkylating and cross-linking agent-induced DNA dam- 1 To whom correspondence should be addressed at LS-6, MS M888, Life Sciences Division, Los Alamos National Lab, Los Alamos, NM age, and in some physiological processes. For example, 87545. Telephone: (505) 667-2789. Fax: (505) 665-3024. E-mail: in the yeast RAD52 group genes result in dchen@.lanl.gov. increased sensitivity to the alkylating agent methyl 2 Abbreviations used: DSB, DNA double-strand break; UBL1, a methanesulfonate (MMS). Indeed, the yeast RAD52 ubiquitin-like protein 1, 12 kDa; Gal4-DA, the DNA activation do- gene was cloned by its ability to complement MMS sen- main (amino acids 768– 881) of Gal-4 protein; Gal4-DB, the DNA binding domain (amino acids 1 –147) of Gal-4 protein; LacZ, b-galac- sitivity (Adzuma et al., 1984; Schild et al., 1983). The tosidase gene; MMS, methyl methanesulfonate. RAD52 gene has been found to function in

271 0888-7543/96 $18.00 Copyright ᭧ 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

AID Genom 4269 / 6r1a$$$361 08-13-96 18:11:20 gnmal AP: Genomics 272 SHEN ET AL. recombination induced by the cross-linking agent pso- ralen (Han and Saffran, 1992; Saffran et al., 1992). In mammalian immune systems, various types of anti- body genes or antigen receptor genes are generated by V(D)J rejoining, where a DNA strand-break is intro- duced, and the DSB repair machinery complies. In other cases, integration of viral DNA into the host ge- nome may require DSB repair-associated mechanisms. During meiosis, mechanisms of chromosome exchange (recombination) may overlap with DSB repair. How- ever, the mechanism of the RAD52-associated repair pathway is poorly understood, especially in human cells. To elucidate the DSB repair mechanism in humans, one of the first steps is to identify human proteins in- volved in DSB repair. It is assumed that some proteins involved in the same repair pathway may associate with each other in the cells. Therefore, one strategy to identify novel proteins participating in the RAD51/ RAD52-dependent repair pathway is to identify pro- teins that actually interact with known proteins, such as RAD51 and RAD52. We have initiated experiments to identify a RAD51/RAD52 interacting protein(s) by using the yeast two-hybrid approach. This approach would also identify a gene(s) involved in meiosis. In this article, we report the cDNA cloning of a ubiquitin- like gene by using the two-hybrid approach. This gene is designated UBL1 for ubiquitin-like 1, as recom- mended by the Human Commit- FIG. 1. Procedures to eliminate false positives in a yeast two- tee. Tissue-specific mRNA expression shows that UBL1 hybrid screen. See text for details. expresses highest in testis. By FISH analysis using a cDNA probe and PCR analysis using a panel of mouse hybrid cells that contain a single human chromosome, The second case is a false positive and was to be eliminated. To do this, the procedure illustrated in Fig. 1 was used. A clone grown on the UBL1 gene was mapped to chromosome 2q32.2– SD/Trp0/Leu0/His0 was grown in SD/Leu0 medium for 5 days and q33; a related sequence may be located on chromosome then plated onto SD/Leu0 agar plates. Due to Try auxotrophs, some 1q23–q25. of the yeast grown on SD/Leu0 agar plates may have lost plasmid pGBT9/RAD51(Trp/). Then, 25 individual colonies from SD/Leu0 plates were duplicately transferred onto SD/Try0/Leu0 and SD/Leu0/ MATERIALS AND METHODS His0 agar plates in an organized manner. When the original clone on SD/Trp0/Leu0/His0 is a true positive, these clones from SD/Leu0 Materials. The yeast strains SFY526 (MATa; ura3-52; his3-200; plates, which do not grow in SD/Trp0/Leu0 (i.e., have lost pGBT9/ ade 2-101; lys 2-801; trp 1-901; leu2-3,112; canr; gal4-542; gal80-538; RAD51), will also not grow in SD/Leu0/His0. If the original clone on URA3::GAL1-LacZ) and HF7c (MATa, ura3-52; his3-200; lys2-801; SD/Trp0/Leu0/His0 plate is a false positive, a colony will grow in SD/ ade2-101; trp1-901; leu2-3,112; gal4-542; gal80-538; LYS2::GAL1- Leu0/His0 even if it has lost pGBT9/RAD51 (i.e., not grown in SD/ 0 0 HIS3; URA3 ::(GAL4 17mers)3-CYC1-LacZ) were purchased from Trp /Leu ). Therefore, true positive clones were identified. The pACT Clontech Laboratories (Palo Alto, CA). SFY526 has a LacZ reporter from positive yeast clones were isolated and electroporated gene fused downstream of the Gal-1 promoter. HF7c has a His re- into HB101 cells, from which a large-scale purification of pACT plas- porter gene controlled by a Gal-1 promoter and a LacZ reporter gene mids was performed. The isolated plasmids were then subjected to controlled by a CYC1 promoter. The Gal-4 DNA activation domain automated DNA sequencing, as will be described later. (Gal4-DA) fused cDNA library in pACT vector was also purchased In vivo assay of protein interaction using the yeast two-hybrid sys- from Clontech. Two hybrid vectors for RAD51 and RAD52 proteins tem. A description of the protein interaction assay using the yeast have been previously reported (Shen et al., 1996a,b). SD and YPD two-hybrid system can be found in the manufacturer’s Matchmaker media/plates were prepared as described in the two-hybrid system manual (Clontech Laboratories) and previous publications (Shen et manual (Clontech Laboratories Inc.). al., 1996a,b). Briefly, plasmids for the two fusion constructs (one Library screen using the yeast two-hybrid system. The library fused with the Gal4-DB, the other fused with Gal4-DA) were cotrans- screening for this yeast two-hybrid system was performed according fected into the genetically constructed yeast cells SFY526. Trans- to the Matchmaker Kit manual (Clontech Laboratory Inc.). Briefly, formed yeast cells were grown on Trp0/Leu0 synthetic agar plates HF7c yeast was first transfected with pGBT9/RAD51 expressing the for 3 days to select yeast clones bearing both fusion vectors. To mea- Gal4-DB/RAD51 fusion protein using the polyethylene glycol/lithium sure the expression of the b-galactosidase (LacZ) reporter gene, acetate method. Yeast HF7c with pGBT9/RAD51 was subsequently which correlates with the interaction of two fusion proteins expressed transfected with cDNAs isolated from the pACT library. These co- from these two vectors, LacZ activity in three independent trans- transfected yeast were grown in SD/Try0/Leu0/His0 agar plates. A formants was measured by filter assay (see Matchmaker Manual; positive clone in SD/Trp0/Leu0/His0 should contain a pACT plasmid and Shen et al., 1996a,b). Quantitative LacZ activity in Miller’s unit coding for a Gal4-DB/RAD51 interacting protein or a protein that is (Miller, 1972) was assayed according to the Matchmaker kit manual able to activate the reporter gene (His) without the RAD51 protein. (Clontech Laboratories). Briefly, yeast from a single clone were grown

AID Genom 4269 / 6r1a$$$361 08-13-96 18:11:20 gnmal AP: Genomics ASSOCIATION OF UBL1 WITH HUMAN RAD51/RAD52 273 overnight in synthetic media lacking Trp/Leu. The density of yeast was determined by measuring the absorbance at 600 nm. Then, 0.1 ml of culture was mixed with 0.7 ml of Z-buffer (16.1 g/liter Na2H- PO4r7H2O; 5.5 g/liter NaH2PO4rH2O; 0.75 g/liter KCl; 0.246 g/liter MgSO4r7H2O; pH 7.0), 50 ml of CHCl3, and 50 ml of 0.1% SDS. O- Nitrophenylgalactoside (4 mg/ml) was used as substrate for LacZ. After 2 h of 30ЊC incubation, the reaction mix was centrifuged, and the absorbance of the supernatant was read at 420 nm. The LacZ in Miller’s unit was calculated as 1000 1 [OD 420/(t 1 V 1 OD 600], where t is time of incubation, V is volume of yeast culture, and OD 600 is the absorbance of yeast culture at 600 nm. Chromosome localization by cDNA FISH mapping. To map the chromosome, the protocol described by Heng et al. was used (Heng et al., 1992; Heng and Tsui, 1993, 1994). Briefly, human lympho- cytes were synchronized with phytohemagglutinin, and metaphase spreads were made by standard procedures. cDNA probe was bio- tinylated with dATP using the BRL BioNick labeling kit and hy- bridized to the chromosome slides. The FISH and DAPI signals were photographed separately. The DAPI chromosome pictures were superimposed on the FISH signal pictures to localize the region better. DNA sequencing. A primer-walking strategy was used to se- quence cloned plasmid cDNA inserts. Sequencing was performed with the Taq DyeDeoxy Terminator Cycle Sequencing Kit and the ABI 373A automatic DNA sequencer (Applied Biosystems Inc., Fos- ter City, CA) according to the manufacturer’s protocols. Both cDNA strands were sequenced at least once. Sequence editing was per- formed with the SeqEd software (Applied Biosystems) on a Macin- tosh computer. Further sequence analysis was performed using the GCG sequence analysis software package (University of Wisconsin, Madison, WI). Northern hybridization. A multiple-tissue Northern blot con- taining mRNA from leukocyte, colon, small intestine, ovary, testis, prostate, thymus, and spleen was purchased from Clontech Labora- FIG. 3. Alignment of the UBL1 amino acid sequence with those of tories. This membrane was sequentially hybridized with UBL1 the yeast SMT3 protein (GenBank Accession No. U33057), ubiquitin, human RAD23A (HHR23A), human RAD23B (HHR23B), and a ubi- quitin cross reacting protein (UCRP). The alignment was performed with the BESTFIT program in the GCG sequence analysis software package (University of Wisconsin, Madison).

cDNA, human RAD51 cDNA, and human b-actin cDNA (provided with the blot), with stripping between each hybridization according to the protocol provided with the membrane. Hybridization probes were labeled with a random labeling kit (Amersham Corp., Arlington Heights, IL). Other molecular methods. Primers were synthesized with an Ap- plied Biosystems Model 394 synthesizer. pGAD424/UBL1 was con- structed by fusing bases 67 –375 (Fig. 2) to the Gal4 DNA activation domain in vector pGAD424, and pGBT424/hRAD12 was constructed by fusing bases 67 –375 (Fig. 2) to the Gal4 DNA binding domain in pGBT9 vector by PCR using BamHI- and SalI-tagged primers. Detailed procedures and construction of other two-hybrid vectors have been described previously (Shen et al., 1996a).

TABLE 1

FIG. 2. cDNA and predicted amino acid sequences. The cDNA Homology between UBL1 and SMT3, Ubiquitin, and clone was isolated by screening a pACT cDNA library (Clontech Labo- Other Ubiquitin-like Proteins ratories) with pGBT9/RAD51 according to the manufacturer’s in- structions and previously described procedures (see Materials and UBL1 SMT3 Ubiquitin HHR23A HHR23B UCRP Methods). His0-selected positive clones were LacZ assayed in HF7c cells using a different promoter. Yeast clone plasmids were isolated UBL1 100% and electroporated into HB101 cells. Growth in M9 (Leu0) minimal SMT3 72% 100% media resulted in selection of containing only the pACT Ubiquitin 45% 43% 100% vector. Purified pACT plasmids were sequenced with an ABI 373A HHR23A 52% 38% 67% 100% automatic DNA sequencer (ABI Biosystems) by using a primer-walk- HHR23B 48% 44% 70% 82% 100% ing strategy. Bidirectional sequencing and at least two independent UCRP 43% 43% 60% 41% 41% 100% sequencing reactions were performed to obtain the cDNA sequence. A potential poly(A) addition signal in the cDNA is underlined. The Note. The BESTFIT program in the GCG package was used for sequence has been deposited with GenBank under Accession No. sequence analysis. Only the N-terminal 85 amino acids were com- U38784. pared for HHR23A and HHR23B.

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FIG. 4. Interaction of UBL1 with RAD51 and RAD52 in a yeast two-hybrid system. (A) Interaction of UBL1 with other proteins by LacZ filter assay. (B) Quantitative LacZ assay for UBL1’s interaction with other proteins. The numbers in parentheses are the average LacZ activity of at least three independent colonies. Error bars indicate the standard errors. Experimental procedures for two-hybrid liquid assay and LacZ unit definition can be found under Materials and Methods. **LacZ activity is statistically higher than that in the controls as tested by Student’s t test (P1 õ 0.001).

RESULTS Comparison of the amino acid sequence with nonre- dundant protein databases (including GenBank, Swiss- cDNA Cloning and Sequence Analysis Port, PIR, etc.) showed no direct match. However, a UBL1 was identified from a pACT vector-based li- yeast protein, SMT3 (GenBank Accession No. U33057), brary through its interaction with the human RAD51 has 72% similarity and 52% identity with UBL1 (Fig. (hRAD51) protein in a two-hybrid system utilizing His 3). Also, many proteins in the ubiquitin family showed and LacZ as selection genes in the HF7c yeast strain moderate homology (40–55% similarity, Ç20% iden- (Clontech Laboratories). Subsequently, a 1017-bp tity) to UBL1. Figure 3 shows the alignment of the cDNA was isolated from 0.3 1 106 independent clones UBL1 protein sequence to human ubiquitin (Callis et (Fig. 2). The cDNA contains an open reading frame al., 1989) and several ubiquitin-like proteins, including starting at base 67 and ending at base 369 (Fig. 2). the N-terminal 85 amino acids of the human RAD23 Because of the characteristic base A at position 64 (Ko- proteins (Masutani et al., 1994; van der Spek et al., zak, 1984), bases 67–69 were assigned as the transla- 1994) and an interferon-inducible protein (UCRP) tion start codon. This open reading frame codes for a (Loeb and Haas, 1992, 1994). Table 1 summarizes the protein of 101 amino acids with a molecular mass of homology between the above-mentioned ubiquitin-like 12 kDa. proteins.

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FIG. 5. mRNA analysis of UBL1. (A) Multiple tissue mRNA blot (Clontech Laboratories) with 2 mg of mRNA on each lane was sequentially hybridized with UBL1 cDNA probe, human RAD51 cDNA probe, and b-actin probe according to the manufacturer’s protocol, with stripping 1 between each hybridization. (B) Northern analysis of UBL1 in human skin fibroblast (HSF) and C3H mouse 102 embryo fibroblast (C3H 1 102 cells). The procedure is as described previously (Shen et al., 1995).

UBL1 Interacts with Human RAD52 as well examined the UBL1 mRNA level in several tissues, as with RAD51 including testis, by Northern blot (Fig. 5A). It is evident that, although UBL1 mRNA is expressed in all the To confirm its interaction with hRAD51, only the cod- tissues tested, testis exhibits the highest level of ing region of UBL1 was fused to the Gal4 DNA activa- mRNA, which is consistent with human RAD51. tion domain in vector pGAD424, which contains a Northern hybridization to human and mouse total weaker promoter than the pACT vector (Clontech Lab- RNA showed a single mRNA species of 1.3–1.4 kb (Fig. oratories). This fusion also eliminated the noncoding 5B), indicating that transcripts of this gene exist in region at the 5؅-end of UBL1 cDNA. UBL1’s interaction human and mouse cells. Since the average poly(A) tail with hRAD51 was further tested in another yeast in an mRNA is about 250 bases (Birnstiel et al., 1985) strain, SFY526 (Clontech Laboratories), using LacZ as and a putative poly(A) addition site (AATAAA) is iden- the reporter gene. As shown in Fig. 4, neither the vector tified in the 1017-bp cDNA clone (Fig. 2), this cDNA alone nor UBL1 itself activated the expression of LacZ. clone (Fig. 2) is at least close to full length. Cotransfection of pVD3 (amino acids 72–390 of fused to the Gal4 DNA binding domain in the pGBT9 vector) or pGBT9/UBL1 with pGAD424/UBL1 did not Chromosome Localization of UBL1 activate the LacZ gene, indicating no association of The chromosome localization of UBL1 was deter- UBL1 with the truncated p53 nor UBL1 itself. How- mined by FISH analysis using the full-length cDNA as ever, when pGBT9/RAD51 or pGBT9/hRAD52 was co- a probe (Heng et al., 1992; Heng and Tsui, 1993, 1994). transfected with pGAD424/UBL1, the LacZ gene was Among 100 cells examined, 50 showed paired chroma- activated, indicating an association of UBL1 with tid signals from chromosome 2q only (Figs. 6A and 6B), hRAD51 as well as hRAD52. 7 showed paired signals from chromosome 1q only, 32 showed signals from both 1q Expression of UBL1 and Human RAD51 mRNA and 2q (Figs. 6C and 6D), and 4 showed paired chroma- in Testis tid signals from (data not shown). There- Since it has been shown that the human RAD51 fore, 82% of the cells showed signals from chromosome mRNA is highly expressed in testis where meiosis and 2q, and 39% of the cells showed signals from chromo- are active (Shinohara et al., 1993), we further some 1q. To define the regional localization further, 10

AID Genom 4269 / 6r1a$$$361 08-13-96 18:11:20 gnmal AP: Genomics FIG. 6. Chromosome localization of the UBL1 gene. (A and B) cDNA FISH (A) and DAPI banding (B) analysis of UBL1 showing the localization on chromosome 2q. (C and D) Signals can be seen from both chromosome 1q and chromosome 2q. See Materials and Methods for technical details.

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partial human (Chen et al., 1994) were used for regional mapping by PCR analysis (Fig. 8B). As shown in Fig. 8B, while DNA from cell lines con- taining the region 2q32.3–q33 (cell lines 6CS-5, XHB- 78, 6x(neo2)-18, and XRV15b(neo2)-11) showed a posi- tive PCR result, cell lines not containing this region (cell lines 6CS-7 and XHB-104) showed negative PCR results. These data confirmed the localization to 2q32.3–q33.

DISCUSSION

Similar to ubiquitin, ubiquitin-like proteins seem to be involved in many cellular processes. For example, a UCRP has been shown to be conjugated to many cellu- lar proteins that are distributed in a cytoskeleton pat- tern. The N-terminal amino acids homologous to ubi- quitin in RAD23 proteins are also essential for RAD23’s DNA repair function (Pejovic, 1995). The location of UBL1 to 2q32.3–q33 and a related gene on 1q23–q24 is worthy of discussion. The 2q33 region has been identified as an aphidicolin-inducible fragile site (Tedeschi et al., 1992), and several cancer cells, such as human small-cell lung carcinoma (Kohno et al., 1994) and ependymomas (Rogatto et al., 1993), have chromosome changes within the region 2q32– FIG. 7. Detailed analysis of 10 individual cells showing the signal q33. A potential tumor suppressor gene has also been localization at 2q32.3– q33 and 1q23– q24 (drawing not to scale). mapped to 1q23–q24 (Horikawa et al., 1995). Genetic changes on 1q23– q25 have been observed in ovarian individual cells were analyzed by comparing the loca- cancers and in -derived cell lines tions of FISH signals with chromosome banding (Fig. (Polito et al., 1995). 7). Based on these data, we concluded that UBL1 is RAD51 shares a moderate and located on 2q32.3–q33 and that a closely related se- some functional similarity (such as DNA binding and quence is located on 1q23–q24. filament formation along DNA strands) with the bacte- To confirm the chromosome localization, from rial recombination protein RecA (Benson et al., 1994; a panel of rodent hybrids, each containing only a single Ogawa et al., 1993; Shinohara et al., 1992; 1993). The human chromosome, were used for PCR analysis as human RAD52 protein shares significant homology described previously (Shen et al., 1995). Figure 8A with the yeast RAD52 protein only at the N-terminus shows that a signal identical to total human DNA was (Muris et al., 1994; Shen et al., 1995). Two independent amplified from human chromosomes 1, 2, and 5 and functional domains involved in self-interaction and in- a weaker signal from , indicating the teraction with RAD51 have been identified (Shen et al., presence of sequences closely related to UBL1 on these 1996a,b). In addition, expression of human RAD52 in chromosomes. monkey cells enhances radiation resistance (Park, We have also searched GenBank for homologous 1995). UBL1’s yeast homolog, SMT3, is a suppressor DNA sequences and found that several expressed se- of the yeast gene MIF2 (see GenBank Acces- quence tags (ESTs) in GenBank have significant nu- sion No. U33057). MIF2 protein is a yeast cleic acid identity with UBL1 cDNA. Two of them (Ac- protein with homology to the mammalian centromere cession Nos. R17443 and T16960) have 99% identity protein CENP-C (Brown, 1995; Meluh and Koshland, with UBL1 in a region of Ç400 bp, presumably because 1995). MIF2 is also involved in yeast chromosome seg- they are ESTs from UBL1 directly. One (GenBank Ac- regation (Brown et al., 1993). These results may there- cession No. T08096) has 62% identity with UBL1 cDNA fore suggest that UBL1 is also involved in mitosis. Co- in a region of 234 bp. Another (GenBank Accession No. incidentally, human RAD51 is highly expressed during T08856) has 57% identity in a region of 184 bp. The the S/G2/M phase of the cell cycle in mammalian cells, last two entries have 81% of the nucleic acids identical and human RAD52 in the G2/M phase (unpublished in a region of 212 bp. Therefore, a family of UBL1 ho- data). We have cloned a human homolog of yeast ubi- mologous genes may exist in the mammalian genome. quitin-conjugation UBC9, which is involved in This may explain why signals from other chromosomes S- and M-phase cyclin degradation (Seufert et al., 1995) are detectable. and mitosis control (Al-Khodairy et al., 1995). This To confirm the regional localization on chromosome 2 hUBC9-like protein also interacts with UBL1, further, a panel of human radiation hybrids containing hRAD52, hRAD51, and p53 proteins (Shen et al.,

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FIG. 8. PCR analysis of human chromosome hybrids in mouse cells as described before. (A) PCR from chromosomes 1, 2, 3, 4, 5, 11, 12, 13, and 14 are shown. ‘‘Buffer’’ denotes negative control using buffer alone as the PCR template, ‘‘mouse A9’’ indicates DNA from mouse A9 cells from which the hybrid panel was constructed, and ‘‘HSF’’ denotes total DNA from human skin fibroblast. Other lanes are molecular size markers. PCR results from other human chromosomes are negative and not shown. The primers used are 5؅GGTGATCAAGCCTCAGTC positions 552– 569 in Fig. 2) and 5؅CCACAGTTCAGTTCTCTG (positions 791–774 in Fig. 2). (B) A few X-ray hybrids, containing partial) human chromosome 2, were further analyzed by PCR. Labels at the top denote the cell lines used; other labels are the same as in A. While DNA from cell lines containing the region 2q32.3 –q33 (cell lines 6CS-5, XHB-78, 6x(neo2)-18, and XRV15b(neo2)-11) showed a positive PCR result, cell lines not containing this region (cell lines 6CS-7 and XHB-104) showed negative PCR results. For detailed information about these cell lines, please refer to Chen et al. (1994).

–1996c). Therefore, we believe the association of UBL1 termination and 3 ؅ processing: The end is in site. Cell 41: 349 with RAD51/RAD52 to be functionally relevant. The 359. association of UBL1 and hRAD51/hRAD52 also sug- Brown, M. T. (1995). Sequence similarities between the yeast chro- gests that DNA repair/recombination pathways and mosome segregation protein Mif2 and the mammalian centromere protein CENP-C. Gene 160: 111– 116. cell cycle/mitosis control pathways may interface with Brown, M. T., Goetsch, L., and Hartwell, L. H. (1993). MIF2 is re- each other. quired for mitotic spindle integrity during anaphase spindle elon- gation in . J. Cell Biol. 123: 387 –403. ACKNOWLEDGMENTS Callis, J., Pollmann, L., Shanklin, J., Wettern, M., and Vierstra, R. (1989). Sequence of a cDNA from Chlamydomonas reinhardii This research was supported by the U.S. Department of Energy encoding a ubiquitin 52 amino acid extension protein. Nucleic and NIH Grant CA50519 to D.J.C. and by a Director’s postdoctoral Acids Res. 17: 8377. fellowship from Los Alamos National Laboratory to Z.S. We thank Chen, D. J., Marrone, B. L., Nguyen, T., Stackhouse, M., Zhao, Y., Drs. Morton E. Bradbury, Scott Peterson, and Robert Cary for helpful and Siciliano, M. J. (1994). Regional assignment of a human DNA discussions and critical reading of the manuscript. repair gene (XRCC5) to 2q35 by X-ray hybrid mapping. Genomics 21: 423– 427. REFERENCES Cole, G. M., Schild, D., and Mortimer, R. K. (1989). Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis. Mol. Cell. Biol. 9: 3101–3104. Adzuma, K., Ogawa, T., and Ogawa, H. (1984). Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol. Cell. Biol. 4: Emery, H. S., Schild, D., Kellogg, D. E., and Mortimer, R. K. (1991). 2735–2744. Sequence of RAD54, a Saccharomyces cerevisiae gene involved in Alani, E., Subbiah, S., and Kleckner, N. (1989). The yeast RAD50 recombination and repair. Gene 104: 103–106. gene encodes a predicted 153-kD protein containing a purine nucle- Firmenich, A. A., Elias-Arnanz, M., and Berg, P. (1995). A novel otide-binding domain and two large heptad-repeat regions. Genet- allele of Saccharomyces cerevisiae RFA1 that is deficient in recom- ics 122: 47–57. bination and repair and suppressible by RAD52. Mol. Cell. Biol. Al-Khodairy, F., Enoch, T., Hagan, I. M., and Carr, A. M. (1995). 15: 1620–1631. The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin Friedberg, E., Siede, W., and Cooper, A. (1991). Cellular response to conjugating enzyme required for normal mitosis. J. Cell Sci. 108: DNA damage in yeast. In ‘‘The Molecular and Cellular Biology of 475–486. the Yeast Saccharomyces’’ (J. Broach, J. R. Pringle, and E. Jones, Basile, G., Aker, M., and Mortimer, R. K. (1992). sequence Eds.), pp. 147 –192, Cold Spring Harbor Laboratory Press, Cold and transcriptional regulation of the yeast recombinational repair Spring Harbor, NY. gene RAD51. Mol. Cell. Biol. 12: 3235–3246. Haaf, T., Golub, E. I., Reddy, G., Radding, C. M., and Ward, D. C. Benson, F. E., Stasiak, A., and West, S. C. (1994). Purification and (1995). Nuclear foci of mammalian Rad51 recombination protein characterization of the human Rad51 protein, an analogue of E. in somatic cells after DNA damage and its localization in synapto- coli RecA. EMBO J. 13: 5764–5771. nemal complexes. Proc. Natl. Acad. Sci. USA 92: 2298– 2302. Birnstiel, M. L., Busslinger, M., and Strub, K. (1985). Han, E. K., and Saffran, W. A. (1992). Differential repair and recom-

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